E6AP蛋白结构域突变真核表达载体的构建及其生物学功能检测  

Construction and characterization of E6AP deletion mutant expression vectors

作　　者：熊加秀 丁丽华[2] 营孙阳[2] 陈志达[3] 张亚楠[2] 贾小萌 陈滢洁 刘文忠 叶棋浓[2] 卫勃[3] 

机构地区：[1]内蒙古医科大学基础医学院,呼和浩特010059 [2]军事医学科学院生物工程研究所,北京100850 [3]解放军总医院普通外科,北京100853 [4]神华神东总医院,陕西榆林719315

出　　处：《军事医学》2016年第2期142-146,共5页Military Medical Sciences

基　　金：国家自然科学基金资助项目(81101883;81272231;31470773);北京市科技新星计划资助项目(2009A38;2011112)

摘　　要：目的构建E6AP蛋白结构域突变真核表达载体,并瞬时转染肿瘤细胞,对其生物学功能进行初步检测。方法以本实验室保存带有myc标签的E6AP质粒为模板,采用PCR技术扩增出E6AP（1～494）结构域和E6AP（495～852）结构域（即E6AP的HECT结构域）编码区（CDS）序列,将其插入到p XJ-40-myc载体中,双酶切和测序验证后瞬时转染人胚肾293T细胞,通过Western印迹鉴定其表达及功能;转染人乳腺癌细胞ZR75-1和人胃癌细胞BGC-823,通过CCK-8法测定细胞生长曲线。结果从E6AP蛋白全长CDS中扩增得到约1479、1071 bp的DNA结构域片段,并成功克隆至p XJ-40-myc载体上,且测序与目的序列完全一致;转染人胚肾293T细胞后,目的蛋白在细胞内成功表达,并且E6AP（495～852）结构域可明显抑制P53蛋白水平,而E6AP（1～494）结构域无抑制作用;细胞生长曲线结果显示,转染E6AP（495～852）结构域突变质粒的乳腺癌、胃癌细胞较空载体细胞生长差异具有统计学意义,而E6AP（1～494）对细胞生长无明显变化。结论成功构建了myc-E6AP（1～494）、（495～852）结构域突变真核表达载体,为进一步研究E6AP蛋白在相关肿瘤发生、发展中的作用奠定一定的基础。Objective To construct deletion mutant eukaryotic expression vectors of E6AP labeled with myc tag and de- tect its activity in cancer cells. Methods E6AP deletion mutants were amplified from myc-E6AP plasmid by PCR and in- serted into the eukaryotic expression vector of pXJ-40-myc. The recombinant plasmids pXJ-40-myc-E6AP（ 1 -494） and （495 -852） were identified by enzyme digestion and sequencing, transfected into HEK293T cells and detected by Western blotting. CCK8 assay was performed to investigate the effect of the deletion mutants on breast cancer and gastric cancer cell proliferation. Results The E6AP deletion mutants of about 1479 and 1071 bp were successfully cloned and expressed. In contrast to E6AP（1-494）, E6AP（495 -852） could inhibit TP53 expression and promote proliferation of ZR75-1 and BGC-823 cells. Conclusion The eukaryotic expression vector of myc-E6AP（ 1 -494）and myc-E6AP（495 -852） is suc- cessfully constructed and expressed, which can facilitate the study of E6AP in related cancer development and progression.

关 键 词：E6AP P53 结构域突变 泛素化 肿瘤 

分 类 号：Q51[生物学—生物化学]